Project Summary/Abstract Shigella flexneri, the causative agent of bacillary dysentery, uses its type III secretion system (T3SS) as a conduit through which effector proteins are shuttled from bacterial to host cell cytoplasm. Once injected, they subvert normal host functions and promote infection and defend against host immune responses. While much about T3SS function has been worked out, details of how the system assembly and activation are controlled are not well understood. Sequence homology between the Shigella protein Spa47 and known ATPases suggests that it may provide the necessary energy through ATP hydrolysis mechanisms. We hypothesized that Shigella use this ATPase to activate and control the T3SS and ultimately the ability to infect human host cells. This proposal describes recent developments that provided the first successful expression and purification of ATPase active Spa47 and biophysical characterization of the purified enzyme. These initial findings provided a strong platform for directed studies showing that Spa47 activation is controlled through the formation of an unprecedented T3SS ATPase homo-trimer that may be driven by the lack of an N-terminal sequence (shared by the Salmonella ATPase InvC) but otherwise conserved amongst T3SS ATPases. Additionally, experiments building on this knowledge identified the Shigella protein MxiN as a potent inhibitor of Spa47 activity, implicating it as a key regulatory component of the Shigella T3SS. To better understand the regulatory role of Spa47 in Shigella T3SS- associated infection and to define the traits of this potentially novel sub-class of T3SS ATPases, the specific aims of this study are to: 1) Use Shigella flexneri as a model system to unravel the relationship between Spa47 oligomerization, ATP hydrolysis activity, and T3SS-associated virulence. and 2) Characterize regulatory interactions between Spa47 and the newly identified Shigella T3SS regulatory protein MxiN. The proposed studies will employ an interdisciplinary approach to directly observe the effects of Spa47 within the context of Shigella as well as determine the influence of oligomerization and MxiN interaction on activation and regulation of the ATPase activity of Spa47. These findings will close a significant gap in the understanding of how an important class of human pathogens including Shigella and Salmonella control their virulence through appropriate timing of T3SS activation. Additionally, these studies will uncover mechanistic details of Spa47 activity that could lead to the development of compounds and methods for more efficiently treating infections by T3SS-expressing pathogens.